Cell wall regeneration by Nicotiana tabacum protoplast: Chemical and biochemical aspects

Abstract

Protoplasts isolated enzymatically from the mesophyll of tobacco leaves (Nicotiana tabacum L.) were cultured in a defined liquid medium. The composition of the cell wall polysaccharides (CWP) and of the extracellular polysaccharides (ECP) synthesized after 1–4 days of culture was investigated by gas-liquid chromatography (GLC) in comparison with that derived from tobacco mesophyll and tobacco tissue cultured on solid agar or in suspension. The cell wall of regenerating protoplasts was composed mainly of non-cellulosic polysaccharides with glucose predominating (65%) and a content of only 5% cellulose whereas the cell walls of leaves and cultured tissue contained 60% and 45% cellulose, respectively. The cell walls of leaves, callus and suspension cultured cells showed differences in their arabinose/ xylose ratio. Galactose, arabinose and uronic acids were the main constituents in the ECP of regenerating protoplast and of suspension cultured cells indicating the secretion of pectic material into the culture medium.
The labelling pattern of polysaccharides synthesized by protoplasts after culture with [U-14C]glucose or [U-14C]sucrose was in accordance with the chemical composition. No label of the generally used osmoticum mannitol was incorporated into polysaccharides. Radioactivity derived from myo-[3H]inositol was transformed into uronic acids and pentoses, but not incorporated into the different hexoses. Simultaneous feeding of [U-14C] glucose and unlabelled myo-inositol reduced incorporation of radioactivity into uronic acids and pentoses. Hence, the myo-inositol pathway is operative in cell wall regenerating tobacco protoplasts.